RNAi MODULATION OF MLL-AF4 AND USES THEREOF

ABSTRACT

The invention relates to compositions and methods for modulating the expression of the MLL-AF4 fusion gene, and more particularly to the downregulation of MLL-AF4 by chemically modified oligonucleotides.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/700,493, filed Feb. 4, 2010, (allowed), which is a divisionalapplication of U.S. application Ser. No. 12/038,808, filed on Feb. 27,2008, now U.S. Pat. No. 7,674,779, issued on Mar. 9, 2010, which is acontinuation application of U.S. application Ser. No. 11/303,367, filedDec. 14, 2005, now U.S. Pat. No. 7,361,752, issued on Apr. 22, 2008,which claims the benefit of U.S. Provisional Application No. 60/635,936,filed Dec. 14, 2004, U.S. Provisional Application No. 60/668,392, filedApr. 5, 2005, and U.S. Provisional Application No. 60/698,414, filedJul. 12, 2005. All the prior applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 15, 2011, is named19429US_CRF_sequencelisting.txt, and is 14,599 bytes in size.

TECHNICAL FIELD

The invention relates to compositions and methods for modulating theexpression of MLL-AF4, and more particularly to the downregulation ofMLL-AF4 by chemically modified oligonucleotides.

BACKGROUND

Chromosomal aberrations giving rise to fusion genes are observed formany different leukemias (Rabbitts, T. H.; Nature 1994; 372: 143-149).Such tumor-specific oncogenes would be promising targets for newtherapeutic approaches with increased specificity, if these oncogeneswere important for maintaining the leukemic phenotype. However, incontrast to the development of a leukemia, a central role for itspersistence has only been established for a minority of leukemic fusiongenes.

The mixed lineage leukaemia (MLL) gene located on chromosome 11q23 isinvolved in numerous chromosomal translocations associated with humanleukemia (Ernst, P., et al.; Curr Opin Hematol 2002; 9:282-287). Themost prevalent among those is the translocation t(4;11)(q21;q23), whichfuses MLL gene with the AF4 gene located on chromosome 4q21 (Gu, Y., etal.; Cell 1992; 71:701-708; McCabe, N. R., et al.; Proc Natl Acad SciUSA 1992; 89:11794-11798; Domer, P. H., et al.; Proc Natl Acad Sci USA1993; 90:7884-7888.). This translocation is the hallmark of a high-riskacute lymphoblastic leukemia (ALL) with a particularly poor prognosis ininfants (Pui, C. H., et al.; Lancet 2002; 359:1909-1915).

The wild-type MLL gene is a member of the trithorax family and encodesfor a 431 kD protein, which is proteolytically processed into twofragments -300 and 180 kD heterodimerizing with each other (Nakamura,T., et al.; Mol Cell 2002; 10:1119-1128; Yokoyama, A., et al.; Blood2002; 100:3710-3718; Hsieh, J. J., et al.; Cell 2003; 115:293-303;Hsieh, J. J., et al.; Mol Cell Biol 2003; 23:186-194.). The MLL proteinhas a complex structure that includes an AT-hook domain for DNA-binding,a MT domain showing homology to DNA methyltransferase (DMT) and methylbinding domain protein 1 (MBD1), a plant homeodomain (PHD) containingzinc fingers and a SET histone methyl transferase domain (Ernst, P., etal.; Curr Opin Hematol 2002; 9:282-287). MLL is involved in mechanismscontrolling hox genes transcription (Ayton, P. M., and Cleary, M. L.;Oncogene 2001; 20:5695-5707.). Interestingly, the Hox genes Hoxa7 andHoxa9 in combination with the homeotic gene Meis-1 are necessary for thetransformation induced by several different MLL fusion genes. Such acrucial role has not yet been reported for MLL-AF4. Nevertheless,expression levels of all three homeotic genes are raised in primaryt(4;11) ALL.

The AF4 gene encodes a serine/proline-rich protein containing nuclearlocalization signal and GTP-binding domain. It localizes to the nucleus(Li, Q., et al.; Blood 1998; 92:3841-3847) and is probably involved intranscriptional activation functions. Whereas the MLL knockout isembryonally lethal (Yu, B. D., et al.; Proc Natl Acad Sci USA 1998;95:10632-10636), AF4-deficient mice exhibit imperfect T-cell developmentand modest alterations in B-cell development (Isnard, P., et al.; Blood2000; 96:705-710).

Notably, the t(4;11) translocation generates two fusion genes, AF4-MLLand MLL-AF4. The significance of either fusion gene for leukemogenesisis not completely understood yet. AF4-MLL has recently been shown tointerfere with ubiquitin-mediated AF4 degradation and to transformmurine embryonic fibroblasts (Bursen, A., et al.; Oncogene 2004;23:6237-6249). Ectopic expression of MLL-AF4 in t(4;11)-negativeleukemic cell lines, however, inhibits cell cycle progression andtriggers apoptosis (Caslini, C., et al.; Leukemia 2004; 18:1064-1071).Paradoxically, 20% of all (4;11) ALL patients lack AF4-MLL either on thetranscriptional or genomic level, whereas MLL-AF4 is always detectabledespite its proapoptotic activities upon ectopic expression (Downing, J.R., et al.; Blood 1994; 83:330-335; Reichel, M., et al.; Oncogene 2001;20:2900-2907). Interestingly, several studies suggest that MLL-AF4fusion oncogene supports cell survival in the t(4;11) context. Cellswith t(4;11) translocation survive extended serum starvation (Kersey, J.H., et al.; Leukemia 1998; 12:1561-1564) and are resistant toCD95-mediated apoptosis (Dörrie, J., et al.; Leukemia 1999;13:1539-1547).

To define the role of this fusion oncogene in leukemogenesis moreprecisely, we applied RNA interference (RNAi) to inhibit MLL-AF4expression in leukemic cells. RNAi is a cellular process resulting inenzymatic cleavage and breakdown of mRNA, guided by sequence-specificdouble-stranded small interfering RNAs (siRNAs) (Dykxhoorn, D. M., etal.; Nat Rev Mol Cell Biol 2003; 4:457-467). Cell transfection withsiRNAs results in the generation of a cytoplasmatically locatedribonucleoprotein complex called RNA-induced silencing complex. Uponactivation of this complex by discarding one of the siRNA strands(Khvorova, A., et al.; Cell 2003; 115:209-216; Schwarz, D. S., et al.;Cell 2003; 115:199-208), the remaining strand targets RISC tocomplementary RNA sequences leading to the endonucleolytic cleavage ofthe target RNA by the RISC component Ago-2 (Meister, G., et al.; MolCell 2004; 15:185-197; Rand, T. A., et al.; Proc Natl Acad Sci USA 2004;101:14385-14389; Song, J. J., et al.; Science 2004; 305:1434-1437).Exogenously added synthetic siRNAs were shown to act as very potent andsequence-specific agents to silence gene expression (Elbashir, S. M., etal.; Nature 2001; 411:494-498), demonstrating the great potential notonly for the analysis of gene function but also for gene-specifictherapeutic approaches (Cheng, J. C., Moore, T. B., and Sakamoto, K. M.;Mol Genet Metab 2003; 80:121-128; Heidenreich, O. Curr Pharm Biotechnol2004; 5:349-354).

In the present study, we used RNAi to specifically inhibit MLL-AF4 geneexpression in t(4;11) cells. We demonstrate that depletion of the fusiontranscript MLL-AF4 inhibits clonogenicity and proliferation, inducesapoptosis in t(4;11)-positive leukemic cells and compromises theirengraftment in a SCID mouse xenotransplantation model.

SUMMARY

The invention provides compositions and methods for reducing MLL-AF4levels in a subject, e.g., a mammal, such as a human. The methodincludes administering to a subject an iRNA agent that reducesexpression of an MLL-AF4 fusion gene (e.g., by at least 2%, 4%, 6%, 10%,15%, 20% or greater) and/or inhibits the rate of proliferation oft(4;11)-positive cells. The iRNA agent can be one described here, or canbe a dsRNA that is based on one of the active sequences and target anidentical region of an MLL-AF4 fusion gene, e.g., a mammalian MLL-AF4fusion gene, such as an MLL-AF4 fusion gene from a human. The iRNA agentcan comprise less than 30 nucleotides per strand, e.g., 21-23nucleotides and consist of, comprise or be derived from one of theagents provided in Table 1, agent numbers 1-12. The double stranded iRNAagent can either have blunt ends or more preferably have overhangs of1-4 nucleotides from one or both 3′ ends of the agent. These preferrediRNA agents preferably include four or more nucleotide mismatches to allnon-MLL-AF4 gene sequences in the subject.

In a first aspect, the invention specifically provides an iRNA agentcomprising a sense strand, wherein the sense strand comprises anucleotide sequence of at least 15 contiguous nucleotides from the sensestrand sequences of agents 1-12 provided in Table 1 (SEQ ID NOs 5, 10,12, 14, 18, 20, 22, 24, 26, 30, and 32), and an antisense strand,wherein the antisense strand comprises at least 15 contiguousnucleotides from the antisense sequences of agents 1-12 provided inTable 1 (SEQ ID NOs 6, 7, 11, 13, 15, 19, 21, 23, 25, 27, 31, and 33),e.g. agent number 5, sense strand sequence 5′-AAGAAAAGCAGACCUACUCCA-3′(SEQ ID NO:14), antisense strand sequence 5′-UGGAGUAGGUCUGCUUUUCUUUU-3′(SEQ ID NO:15). The iRNA agents of Table 1, agent numbers 1-12, possessthe advantageous and surprising ability to reduce the amount of MLL-AF4mRNA present in cultured human SEM cells (leukemia cell line) afterincubation with these agents by more than 40% compared to cells whichhave not been incubated with the agent (see FIG. 1).

In a second aspect, the invention provides an iRNA agent comprising anucleotide sequence in the sense strand and a nucleotide sequence in theantisense strand each comprising a sequence of at least 16, 17 or 18nucleotides which is essentially identical to one of the sequences ofagents 1-12 of Table 1 (sense strand: SEQ ID NOs 5, 10, 12, 14, 18, 20,22, 24, 26, 30, and 32; antisense strand: SEQ ID NOs 6, 7, 11, 13, 15,19, 21, 23, 25, 27, 31, and 33), except that not more than 1, 2 or 3nucleotides per strand, respectively, have been substituted by othernucleotides (e.g. adenosine replaced by uracil), while essentiallyretaining the ability to inhibit MLL-AF4 expression in cultured humanSEM cells.

The iRNA agents of the invention may comprise a sense strand comprisingat least 15 contiguous nucleotides from the sense strand sequences ofagents 1, 2, 5, and 9 provided in Table 1 (SEQ ID NOs 5, 14, and 24),and an antisense strand comprising at least 15 contiguous nucleotides ofthe antisense sequences of agents 1, 2, 5, and 9 provided in Table 1(SEQ ID NOs 6, 7, 15, and 25), wherein the iRNA agents reduce the amountof MLL-AF4 mRNA present in cultured human SEM cells after incubationwith these agents by more than 60% compared to cells which have not beenincubated with the agent. The antisense strand of the iRNA agents of theinvention may be 30 or fewer nucleotides in length, and the duplexregion of the iRNA agents may be 15-30 nucleotide pairs in length. TheiRNA agents may comprise at least one nucleotide overhang having 1 to 4unpaired nucleotides, preferably 2 or 3 unpaired nucleotides. Thenucleotide overhang may be at the 3′-end of the antisense strand of theiRNA agent.

Furthermore, the iRNA agents of the invention may consist of

(a) a double stranded structure formed by a sense strand having anucleotide sequence chosen from the group of: the sequence ofnucleotides of from position (5′ to 3′) 1 to 19, or 1 to 21, of thesense strand sequences SEQ ID NOs 5, 10, 12, 14, 18, 20, 22, 24, 26, 30,and 32, and an antisense strand having a nucleotide sequence chosen fromthe group of: the sequence of nucleotides of from positions (5′ to 3′) 1to 21, or 3 to 21, of the antisense strand sequences SEQ ID NOs 6, 11,13, 15, 19, 21, 23, 25, 27, 31, and 33, if the sense strand is chosen tohave a sequence of nucleotides of from positions 1 to 21 of said sensestrand sequences, and the sequence of nucleotides of from positions 3 to21 of the antisense strand sequences SEQ ID NOs 6, 11, 13, 15, 19, 21,23, 25, 27, 31, and 33, if the sense strand is chosen to have a sequenceof nucleotides of from positions 1 to 19 of said sense strand sequences,

(b) at least one single stranded overhang of 1 to 4 unpaired nucleotidesat the 3′-end of the sense and/or antisense strand, wherein said singlestranded overhang, if at the 3′-end of the sense strand, optionallycomprises the nucleotides in positions 20 and 21 of the said sensestrand sequences in their respective positions, and/or, if at the 3′-endof the antisense strand, optionally comprises the nucleotides inpositions 22 and 23 of the said antisense strand sequences in theirrespective positions,

wherein the iRNA agent does not comprise a single stranded overhang of 1to 4 unpaired oligonucleotides on the 3′-end of the sense strand, if thedouble stranded structure is formed by a sense strand having a sequenceof nucleotides of from position 1 to 21 of the said sense strandsequences and an antisense strand having a sequence of nucleotides offrom position 1 to 21 of said antisense strand sequences. Specifically,the nucleotide sequence of the sense strand may consist of thenucleotide sequence of SEQ ID NO:14, and the nucleotide sequence of theantisense strand may consist of the nucleotide sequence of SEQ ID NO:14.

The iRNA agents of the invention are meant to reduce the amount ofMLL-AF4 mRNA present in cultured human SEM cells after incubation withthe iRNA agent by more than 40% compared to cells which have not beenincubated with the agent. They may further comprise a modification thatcauses the iRNA agent to have increased stability in a biologicalsample, which may be a phosphorothioate or a 2′-modified nucleotide. Forexample, the iRNA agents may comprise at least one 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide;at least one 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide; at least one5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide. The 2′-modification may be chosen from the group of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).

The iRNA agents may further comprise a cholesterol moiety, which ispreferably conjugated to the 3′-end of the sense strand of the iRNAagent. The iRNA agents may be targeted for uptake by cells of the liveror by cells of the gut.

In a third aspect, the invention provides a pharmaceutical composition,comprising:

(a) an iRNA agent of the invention; and

(b) a pharmaceutically acceptable carrier.

The pharmaceutical composition may further comprise a formulating agentwhich prolongs the half-life of the iRNA agent in human and/or mouseserum, or which facilitates uptake of the iRNA agent into cells. Theformulating agent may comprise a polycation. Preferably, suchpharmaceutical composition is useful in the treatment of a proliferativedisorder e.g. cancer, preferably leukemia, more preferably acutelymphoblastic leukemia. Preferably, the pharmaceutical composition iscapable of reducing MLL-AF4 fusion gene expression in a cell or tissueof a subject (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20% or greater)and/or of inhibiting the rate of proliferation of t(4;11)-positive cellsupon administration of the pharmaceutical composition to the subject,e.g. a human.

In a fourth aspect, the invention provides a method of treating a humansubject having or at risk for developing a proliferative disorder,comprising the step of: administering an iRNA agent of the invention tothe human subject. The proliferative disorder is preferably cancer, andmore preferably acute lymphoblastic leukemia. Therein, the iRNA agentmay be administered in an amount sufficient to reduce the expression ofMLL-AF4 in a cell or tissue of the subject (e.g., by at least 2%, 4%,6%, 10%, 15%, 20% or greater) and/or of inhibiting the rate ofproliferation of t(4;11)-positive cells.

In a fifth aspect, the invention provides a method of making apharmaceutical composition, comprising the step of: formulating one ofthe iRNA agents of the invention with a pharmaceutical carrier, andoptionally further comprises the step of: formulating the iRNA agentwith a formulating agent which prolongs the half-life of the iRNA agentin human and/or mouse serum, or which facilitates uptake of the iRNAagent into cells. The formulating agent may comprise a polycation. Thepharmaceutical composition of the inventive method is preferably usefulin the treatment of a proliferative disorder, which is preferablycancer, more preferably a leukemia, and most preferably acutelymphoblastic leukemia. In one preferred embodiment, the pharmaceuticalcomposition of the inventive method is capable of reducing MLL-AF4fusion gene expression in a cell or tissue of a subject (e.g., by atleast 2%, 4%, 6%, 10%, 15%, 20% or greater) and/or of inhibiting therate of proliferation of t(4;11)-positive cells upon administration ofthe pharmaceutical composition to the subject, e.g. a human.

In a sixth aspect, the invention provides a method of reducing theamount of MLL-AF4 RNA in a cell (e.g., by at least 2%, 4%, 6%, 10%, 15%,20% or greater) and/or of inhibiting the rate of proliferation oft(4;11)-positive cells, comprising the step of: contacting the cell withan iRNA agent of the invention. In one embodiment, the method isperformed in vitro. Alternatively, the cell is a cell of a subject, andthe subject is diagnosed as having a proliferative disorder, preferablycancer, and more preferably acute lymphoblastic leukemia. The cells canbe located in a tumor, or they can be microclonal cells.

In a seventh aspect, the invention provides a method of making an iRNAagent of the invention, the method comprising the step of: synthesizingthe sense and the antisense strand of the iRNA agent, wherein the senseand/or antisense strands comprise at least one nucleotide modification;said nucleotide modification is preferably a modification that causesthe iRNA agent to have increased stability in a biological sample. Saidmethod optionally further comprises the step of: administering the iRNAagent to a subject. In one embodiment, the subject is diagnosed ashaving a proliferative disorder, preferably cancer, and more preferablyacute lymphoblastic leukemia. Therein, the subject may be a human.

TABLE 1 MLL-AF4 siRNA sequences^(d). Agent Strand SEQ ID No: ID:Sequence^(c) NO: 1 MA3S^(a)   5′-AAAAG/CAGACCUACUCCAAUG-3′ 5 MA3as^(b)3′-UCUUUUC/GUCUGGAUGAGGUUAC-5′ 6 2 MA3s   5′-AAAAG/CAGACCUACUCCAAUG-3′ 5MAxas 3′-UCUUUUC/GUCUGGAUGAGGUU-5′ 7 3 MA4s  5′-GAAAAG/CAGACCUACUCCAAU-3′ 10 MA4as 3′-UUCUUUUC/GUCUGGAUGAGGUUA-5′11 4 MA5s   5′-AGAAAAG/CAGACCUACUCCAA-3′ 12 MA5as3′-UUUCUUUUC/GUCUGGAUGAGGUU-5′ 13 5 MA6s   5′-AAGAAAAG/CAGACCUACUCCA-3′14 MA6as 3′-UUUUCUUUUC/GUCUGGAUGAGGU-5′ 15 6 MA8s  5′-AAAAGAAAAG/CAGACCUACUC-3′ 18 MA8as 3′-GGUUUUCUUUUC/GUCUGGAUGAG-5′19 7 MA9s   5′-CAAAAGAAAAG/CAGACCUACU-3′ 20 MA9as3′-UGGUUUUCUUUUC/GUCUGGAUGA-5′ 21 8 MA10s   5′-CCAAAAGAAAAG/CAGACCUAC-3′22 MA10as 3′-UUGGUUUUCUUUUC/GUCUGGAUG-5′ 23 9 MA11s  5′-ACCAAAAGAAAAG/CAGACCUA-3′ 24 MA11as 3′-UUUGGUUUUCUUUUC/GUCUGGAU-5′25 10 MA12s   5′-AACCAAAAGAAAAG/CAGACCU-3′ 26 MA12as3′-UUUUGGUUUUCUUUUC/GUCUGGA-5′ 27 11 MA14s  5′-AAAACCAAAAGAAAAG/CAGAC-3′ 30 MA14as 3′-GUUUUUGGUUUUCUUUUC/GUCUG-5′31 12 siMARS   5′-ACUUUAAGCAGACCUACUCCA-3′ 323′-CCUGAAAUUCGUCUGGAUGAGGU-5′ 33 ^(a)“s” indicates sense strand ^(b)“as”indicates antisense strand ^(c)“/” indicates the fusion betweenchromosome 11 and chromosome 4 sequence ^(d)duplexed siRNA agents arereferenced in the specification as “siMAxx”

In an eighth aspect, the invention provides a method of evaluating aniRNA agent thought to inhibit the expression of an MLL-AF4 gene, themethod comprising:

-   -   (a) providing an iRNA agent, wherein a first strand is        sufficiently complementary to a nucleotide sequence of an        MLL-AF4 mRNA, and a second strand is sufficiently complementary        to the first strand to hybridize to the first strand;    -   (b) contacting the iRNA agent to a cell comprising an MLL-AF4        gene;    -   (c) comparing MLL-AF4 gene expression before contacting the iRNA        agent to the cell, or of uncontacted control cells, to the        MLL-AF4 gene expression after contacting the iRNA agent to the        cell; and    -   (d) determining whether the iRNA agent is useful for inhibiting        MLL-AF4 gene expression, wherein the iRNA is useful if the        amount of MLL-AF4 RNA present in the cell, or protein secreted        by the cell, is less than the amount present or secreted prior        to contacting the iRNA agent to the cell, or less than the        amount present or secreted by cells not so contacted.

In one embodiment of this method, the iRNA agent is an iRNA agent of theinvention as described above.

The iRNA agents can either contain only naturally occurringribonucleotide subunits, or can be synthesized so as to contain one ormore modifications to the sugar or base of one or more of theribonucleotide subunits that is included in the agent. The iRNA agentcan be further modified so as to be attached to a ligand that isselected to improve stability, distribution or cellular uptake of theagent, e.g. cholesterol. The agents can further be in isolated form orcan be part of a pharmaceutical composition used for the methodsdescribed herein.

In another embodiment, and as described herein, a cholesterol moiety(e.g., on the 3′-end of the sense strand), a 2′-modification (e.g., a2′-O-methyl or 2′-deoxy-2′-fluoro-modification), and a phosphorothioate(e.g., on the 3′-most one or two nucleotides of the sense and antisensestrands) are present in the same iRNA agent.

Preferably, administration of an iRNA agent, e.g., an iRNA agentdescribed herein, is for treatment of a disease or disorder present inthe subject in which MLL-AF4 fusion gene expression plays a role.Administration of the iRNA agent may also be performed for prophylactictreatment of disorders mediated by the MLL-AF4 fusion.

The invention features preparations, including substantially pure orpharmaceutically acceptable preparations of iRNA agents which modulatee.g., inhibit, MLL-AF4. The iRNA agent that targets MLL-AF4 can beadministered to a subject, wherein the subject is at risk for developingor having (e.g., diagnosed as having) a disorder characterized by thepresence of the MLL-AF4 fusion gene, or other t(4;11) chromosomaltranslocations. The iRNA agent can be administered to an individualdiagnosed with or having the disorder, or at risk for the disorder todelay onset of the disorder or a symptom of the disorder. Thesedisorders include proliferative disorders, such as t(4;11)-associatedleukemias, including acute lymphoblastic leukemias. For example, theiRNA agent that targets MLL-AF4 may be administered to a subject having(e.g., diagnosed as having) infant acute lymphoblastic leukemia,leucocytosis or an extramedullary disease in one or more organs(formation of blood cells outside of the bone marrow), e.g., the spleen,liver or lymph nodes.

The iRNA agent can also be targeted to a specific tissue such as thebone marrow, and MLL-AF4 expression levels in the tissue (e.g., in atumor of the tissue) are decreased following administration of theMLL-AF4 iRNA agent. Preferably, the iRNA agent is modified to maximizethe time the iRNA agent spends in the blood stream. For example, theiRNA agent can be associated with human serum albumin (HSA).

In the methods and compositions of the invention, the iRNA agent may bemodified, or associated with a delivery agent, e.g., a delivery agentdescribed herein, e.g., a liposome. Such modification may mediateassociation with a serum albumin (SA), e.g., a human serum albumin(HSA), or a fragment thereof, to increase the circulation time of theagent.

The present agents, methods and compositions utilize the cellularmechanisms involved in RNA interference to selectively degrade MLL-AF4fusion mRNA in a cell. Therefore, the inventive methods will usuallycomprise a step of contacting a cell with one of the iRNA agents of thepresent invention. Such methods can be performed directly on a cell orcan be performed on a mammalian subject by administering to a subjectone of the iRNA agents of the present invention. In preferredembodiments, the cells are contacted with the iRNA agent at least twice,preferably at intervals of 48 hours. Reduction of MLL-AF4 fusion mRNA ina cell results in a reduction in the amount of MLL-AF4 fusion proteinproduced, and in an organism, may result in a decrease in MLL-AF4 fusionmediated disease effects.

The methods and compositions featured in the invention, e.g., themethods and compositions to treat the proliferative disorders describedherein, can be used with any of the iRNA agents described. In addition,the methods and compositions featured in the invention can be used forthe treatment of any disease or disorder described herein, and for thetreatment of any subject, e.g., any animal, any mammal, such as anyhuman.

The methods and compositions featured in the invention, e.g., themethods and iRNA compositions to treat the proliferative disordersdescribed herein, can be used with any dosage and/or formulationdescribed herein, as well as with any route of administration describedherein.

The details of one or more embodiments featured in the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthis description, the drawings, and from the claims. This applicationincorporates all cited references, patents, and patent applications byreferences in their entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1F. Activity and specificity of MLL-AF4 siRNAs. FIG. 1A: siRNAscan of the MLL-AF mRNA fusion site. MLL-AF4 mRNA levels normalized byGAPDH mRNA levels are shown. Target sites of the indicated siRNAs weremoved by one single nucleotide from the AF4 part to the MLL part of thefusion site. Total RNA was isolated 24 h after electroporation with 500nM of siRNA and analyzed by real time RT-PCR. Numbers on x-axiscorrespond to numbering of siRNAs in Table 2 (siMAn, n=1 to 14). Errorbars show standard deviations. FIG. 1B: Time course of MLL-AF4depletion. Total RNA was isolated at the indicated time points aftermock electroporation, or electroporation with 750 nM siRNA siMA6 or themismatch control siMAmm. Real time RT-PCR was performed as in FIG. 1A.FIG. 1C: Effects of the MLL-AF4 siRNA siMA6 and a mismatch control siMMon MLL-AF4, AF4 and MLL mRNA levels in SEM cells. Analysis was performedas in FIG. 1A. FIG. 1D: Depletion of MLL-AF4 protein upon siRNAtransfection. Total cell lysates were isolated 48 h after mockelectroporation, or electroporation with 500 nM siRNA siMA6 or mismatchcontrol siMAmm. MLL-AF4 was detected with an antibody targeting theC-terminus of AF4. GAPDH served as a loading control and fornormalization. Normalized MLL-AF4 protein levels are indicated at thebottom. FIG. 1E Effects of the MLL-AF4 siRNAs siMA6 and siMARS and amismatch control siMM on MLL-AF4, AF4 and MLL mRNA levels in RS4;11cells. Analysis was performed as in FIG. 1A. siMA6 is homologous to theMLL-AF4 variant expressed in SEM cells, siMARS targets the variantpresent in RS4;11 cells. FIG. 1F: MLL-AF4 siRNAs do not induce aninterferon response. SEM cells were transfected with the indicated RNAs.PolyIC (7.5 μg/mg) served as a positive control for the induction of theinterferon response genes OAS1 and STAT1. Analysis was performed as inFIG. 1A.

FIGS. 2A-2E. MLL-AF4 depletion inhibits colony formation andproliferation of t(4;11)-positive leukemic cells. FIG. 2A: Specificityof MLL-AF4 and AML1/MTG8 siRNAs. SEM cells express MLL-AF4, whereasKasumi-1 cells express AML1/MTG8. The number of colonies formed bysiMA6-treated SEM or siAGF1-treated Kasumi-1 cells are significantlylower than those of cells treated with the respective mismatch controls(p<0.001). FIG. 2B: Inhibition of RS4;11 clonogenicity is dependent onperfect homology to the MLL-AF4 fusion site. The number of coloniesformed by siMARS treated RS4;11 cells is significantly lower than bymock-treated RS4;11 cells, RS4;11 cells treated with thee9-e4-variant-specific siMA6, or the e9-e4 mismatch control siMAmm(p<0.0001). FIG. 2C: siRNAs targeting MLL-AF4 do not affect colonyformation of primary human CD34+ hemopoetic cells. Primary human CD34+hemopoetic cell were electroporated with 750 nM siMA6 (siRNA targetingMLL-AF4 e9-e4 variant expressed in SEM cells), siMARS (siRNA targetingMLL-AF4 e10-e4 variant expressed in RS4;11 cells), or the e9-e4 mismatchcontrol siMM. Error bars show standard deviations. FIG. 2D: Growthcurves of siRNA-treated SEM and RS4;11 cells. Every second day, cellswere mock electroporated or electroporated with 750 nM siMA6, siMARS, orthe mismatch control siMAmm. Cell numbers were determined using the MTTassay. Only the siRNA having complementarity to the fusion site variantpresent in the respective cell line was able to suppress growth. Errorbars indicate standard deviations. FIG. 2E: Effects of MLL-AF4 siRNAs onthe cell cycle distribution of SEM and RS4;11 cells. The graphs show thepercentage of cells in the indicated cycle phase. Cell cycledistribution was determined by flow cytometry at the indicated daysusing cells from the time course experiments shown in FIG. 2D.

FIGS. 3A-3C. MLL-AF4 depletion induces apoptosis in t(4;11)-positive SEMand RS4;11 cells. FIG. 3A: Effects of MLL-AF4 suppression on thefraction of sub G1 cells. Cells obtained from the time courses shown inFIGS. 2D and 2E were analyzed for DNA content by flow cytometry. FIG.3B: Annexin V staining of SEM cells Annexin V-positive SEM cells werequantified by flow cytometry 4 days after the second electroporationwith 750 nM of the indicated siRNA. The percentages of annexin V andannexin V/propidium iodide-positive cells are given in the correspondingquadrants. FIG. 3C: MLL-AF4 suppression triggers caspase-3 activationand diminishes Bcl-XL protein levels. Immunoblots show proteolyticallyactivated caspase-3 and Bcl-XL proteins. Tubulin and GAPDH served asloading controls.

FIG. 4. MLL-AF4 suppression affects Hoxa7 and Hoxa9 gene expression.Total RNA was isolated 48 h after the second electroporation with 500 nMof the indicated siRNA and analyzed by real time RT-PCR. Error bars showstandard deviations.

FIGS. 5A-5E. MLL-AF4 suppression diminishes leukemic engraftment. FIG.5A: Survival curves of SCID mice transplanted with SEM cells. Prior totransplantation, SEM cells were either mock electroporated twice, orelactroporated twice with siRNAs siMA 6 or its mismatch control siMAmm.Pretreatment with the MLL-AF4 e9-e4-specific siRNA siMA6 extended mediansurvival and increased overall survival significantly compared to mockor control siRNA pretreatment (p<0.01 according to log-rank test). FIG.5B: FACS analysis of bone marrow. Bone marrow cells of animals werestained with anti-human CD45 antibody and analyzed by flow cytometry.Animals treated with siMA6 had considerably lower CD45 positive cellcounts. FIG. 5C: Liver and spleen histologies. Original magnification200×; scale bar, 50 μm. Mice transplanted with mock or siMM-pretreatedcells were moribund at the time of analysis. The animal transplantedwith siMA6-pretreated cells was sacrificed 228 days aftertransplantation without any sign of leukemia-associated morbidity. FIG.5D: Comparison of spleen size. The spleen on the left stems from a mocktreated animal, the spleen in the middle from an animal treated with themismatch control siRNA siMM, the right spleen from an animal treatedwith siRNA siMA6 specific for MLL-AF4. FIG. 5E: Graphical representationof organ weights. Organ weights were normalized to whole body weight.Normalized liver and spleen weights of surviving animals of the siMA6group were significantly smaller than those from the mock or siMMmismatch control group (p<0.05 and p<0.001, respectively).

DETAILED DESCRIPTION

For ease of exposition the term “nucleotide” or “ribonucleotide” issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood that the usage of the term“ribonucleotide” or “nucleotide” herein can, in the case of a modifiedRNA or nucleotide surrogate, also refer to a modified nucleotide, orsurrogate replacement moiety, as further described below, at one or morepositions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are described herein. While numerousmodified RNAs and nucleoside surrogates are described, preferredexamples include those which have greater resistance to nucleasedegradation than do unmodified RNAs. Preferred examples include thosethat have a 2′ sugar modification, a modification in a single strandoverhang, preferably a 3′ single strand overhang, or, particularly ifsingle stranded, a 5′-modification which includes one or more phosphategroups or one or more analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can downregulate the expression of atarget gene, such as the target fusion gene, MLL-AF4. While not wishingto be bound by theory, an iRNA agent may act by one or more of a numberof mechanisms, including post-transcriptional cleavage of a target mRNAsometimes referred to in the art as RNAi, or pre-transcriptional orpre-translational mechanisms. An iRNA agent can be a double strandediRNA agent.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interstrand hybridization can form aregion of duplex structure. A “strand” herein refers to a contiguoussequence of nucleotides (including non-naturally occurring or modifiednucleotides). The two or more strands may be, or each form a part of,separate molecules, or they may be covalently interconnected, e.g. by alinker, e.g. a polyethyleneglycol linker, to form but one molecule. Atleast one strand can include a region which is sufficientlycomplementary to a target RNA. Such strand is termed the “antisensestrand”. A second strand comprised in the dsRNA agent which comprises aregion complementary to the antisense strand is termed the “sensestrand”. However, a ds iRNA agent can also be formed from a single RNAmolecule which is, at least partly; self-complementary, forming, e.g., ahairpin or panhandle structure, including a duplex region. In such case,the term “strand” refers to one of the regions of the RNA molecule thatis complementary to another region of the same RNA molecule.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and/or host (Manche, L., et al., Mol.Cell. Biol. 1992, 12:5238; Lee. S B, Esteban, M, Virology 1994, 199:491;Castelli, J C, et al., J. Exp. Med. 1997, 186:967; Zheng, X.,Bevilacqua, P C, RNA 2004, 10:1934; Heidel et al., Nature Biotechn.2004, 22:1579). The iRNA agents of the present invention includemolecules which are sufficiently short that they do not trigger adeleterious non-specific interferon response in normal mammalian cells.Thus, the administration of a composition of an iRNA agent (e.g.,formulated as described herein) to a subject can be used to silenceexpression of the MLL-AF4 fusion gene in MLL-AF4 fusion expressing cellscomprised in the subject, while circumventing an interferon response.Molecules that are short enough that they do not trigger a deleteriousinterferon response are termed siRNA agents or siRNAs herein. “siRNAagent” or “siRNA” as used herein, refers to an iRNA agent, e.g., a dsiRNA agent, that is sufficiently short that it does not induce adeleterious interferon response in a human cell, e.g., it has a duplexedregion of less than 60 but preferably less than 50, 40, or 30 nucleotidepairs.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents, can mediate silencing of an MLL-AF4 fusion gene, e.g., byRNA degradation. For convenience, such RNA is also referred to herein asthe RNA to be silenced. Such a gene is also referred to as a targetgene. Preferably, the RNA to be silenced is a gene product of an MLL-AF4gene fusion that is endogenous to the cell, e.g. a leukemic cell.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.

As used herein, “the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound featured in the invention and a targetRNA molecule, e.g. an MLL-AF4 mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least four nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target MLL-AF4 mRNA) if theiRNA agent reduces the production of a protein encoded by the target RNAin a cell. The iRNA agent may also be “exactly complementary” (excludingthe SRMS containing subunit(s)) to the target RNA, e.g., the target RNAand the iRNA agent anneal, preferably to form a hybrid made exclusivelyof Watson-Crick basepairs in the region of exact complementarity. A“sufficiently complementary” iRNA agent can include an internal region(e.g., of at least 10 nucleotides) that is exactly complementary to atarget MLL-AF4 RNA. Moreover, in some embodiments, the iRNA agentspecifically discriminates a single-nucleotide difference. In this case,the iRNA agent only mediates RNAi if exact complementary is found in theregion (e.g., within 7 nucleotides of) the single-nucleotide difference.Preferred iRNA agents will be based on or consist or comprise the senseand antisense sequences provided in Table 1.

As used herein, “essentially identical” when used referring to a firstnucleotide sequence in comparison to a second nucleotide sequence meansthat the first nucleotide sequence is identical to the second nucleotidesequence except for up to one, two or three nucleotide substitutions(e.g., adenosine replaced by uracil). “Essentially retaining the abilityto inhibit MLL-AF4 expression in cultured human SEM cells” as usedherein referring to an iRNA agent not identical to but derived from oneof the iRNA agents of Table 1 by deletion, addition or substitution ofnucleotides, means that the derived iRNA agent possesses an inhibitoryactivity lower by not more than 20% inhibition compared to the iRNAagent of Table 1 it was derived from. For example, an iRNA agent derivedfrom an iRNA agent of Table 1, which lowers the amount of MLL-AF4 mRNApresent in cultured human SEM cells by 40% may itself lower the amountof MLL-AF4 mRNA present in cultured human SEM cells by at least 40% inorder to be considered as essentially retaining the ability to inhibitMLL-AF4 expression in cultured human SEM cells. Optionally, an iRNAagent featured in the invention may lower the amount of MLL-AF4 mRNApresent in cultured human SEM cells, by at least 40%.

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder mediated by MLL-AF4 fusion protein expression.The subject can be any mammal, such as a cow, horse, mouse, rat, dog,pig, goat, or a primate. In the preferred embodiment, the subject is ahuman.

As used herein, disorders associated with MLL-AF4 fusion expressionrefers to any biological or pathological state that 1) is mediated inpart by the presence of MLL-AF4 fusion protein and 2) whose outcome canbe affected by reducing the level of MLL-AF4 fusion protein present.Specific disorder associated with MLL-AF4 fusion expression are notedbelow.

Disorders Associated with MML-AF4 Misexpression

The MLL-AF4 fusion product has been associated with acute lymphoblasticleukemias (ALLs), such as infant acute lymphoblastic leukemia. Patientshaving an ALL typically present marked leucocytosis and extramedullarydisease in multiple organs, respond poorly to chemotherapy and have poorprognosis.

Design and Selection of iRNA Agents

The present invention is based on the demonstration of silencing of anMLL-AF4 fusion gene in vitro in cultured cells after incubation with aniRNA agent, and the resulting antiproliferative effect.

An iRNA agent can be rationally designed based on sequence informationand desired characteristics. For example, an iRNA agent can be designedaccording to the relative melting temperature of the candidate duplex.Generally, the duplex should have a lower melting temperature at the 5′end of the antisense strand than at the 3′ end of the antisense strand.

Candidate iRNA agents can also be designed by performing, for example, agene walk analysis of the genes that will serve as the target gene.Overlapping, adjacent, or closely spaced candidate agents correspondingto all or some of the transcribed region can be generated and tested.Each of the iRNA agents can be tested and evaluated for the ability todown regulate the target gene expression (see below, “Evaluation ofCandidate iRNA agents”).

Example 1 herein below shows a gene walk approach was used to evaluatepotential iRNA agents targeting the fusion site of human MLL-AF4 mRNA.Based on the results provided, Table 1 provides active iRNA agentstargeting MLL-AF4. As shown in the Examples below, the iRNA agents ofTable 1, agent numbers 1-12, possess the advantageous and surprisingability to reduce the amount of MLL-AF4 fusion mRNA present in culturedMLL-AF4 fusion gene expressing cells after incubation with these agentsby more than 40% compared to cells which have not been incubated withthe agent.

Based on these results, the invention specifically provides an iRNAagent that includes a sense strand having at least 15 contiguousnucleotides of the sense strand sequences of the agents provided inTable 1, and an antisense strand having at least 15 contiguousnucleotides of the antisense sequences of the agents provided in Table 1under agent numbers 1-12.

The iRNA agents shown in Example 1 hereinbelow, except for agent number2, are composed of a sense strand of 21 nucleotides in length, and anantisense strand of 23 nucleotides in length. However, while theselengths may potentially be optimal, the iRNA agents are not meant to belimited to these lengths. The skilled person is well aware that shorteror longer iRNA agents may be similarly effective, since, within certainlength ranges, the efficacy is rather a function of the nucleotidesequence than strand length. For example, Yang, D., et al., PNAS 2002,99:9942-9947, demonstrated similar efficacies for iRNA agents of lengthsbetween 21 and 30 base pairs. Others have shown effective silencing ofgenes by iRNA agents down to a length of approx. 15 base pairs (Byrom,W. M., et al., Inducing RNAi with siRNA Cocktails Generated by RNaseIII; Tech Notes 10(1), Ambion, Inc., Austin, Tex., USA).

Therefore, it is possible and contemplated by the instant invention toselect from the sequences provided in Table 1 a partial sequence ofbetween 15 to 22 nucleotides for the generation of an iRNA agent derivedfrom one of the sequences provided in Table 1. Alternatively, one mayadd one or several nucleotides to one of the sequences provided in Table1, preferably, but not necessarily, in such a fashion that the addednucleotides are complementary to the respective sequence of the targetgene, e.g. an MLL-AF4 fusion gene. All such derived iRNA agents areincluded in the iRNA agents of the present invention, provided theyessentially retain the ability to inhibit MLL-AF4 expression in culturedhuman SEM cells.

The antisense strand of an iRNA agent should be equal to or at least,14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. Itshould be equal to or less than 60, 50, 40, or 30, nucleotides inlength. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The sense strand of an iRNA agent should be equal to or at least 14, 15,16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should beequal to or less than 60, 50, 40, or 30 nucleotides in length. Preferredranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength.

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50nucleotide pairs in length. It should be equal to or less than 60, 50,40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to25, 19 to 23, and 19 to 21 nucleotides pairs in length.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the MLL-AF4 fusion gene, and are ofsufficient length in terms of nucleotides, that the iRNA agent, or afragment thereof, can mediate down regulation of the MLL-AF4 fusiongene. The antisense strands of the iRNA agents of Table 1, are fullycomplementary to the mRNA sequences of human MLL-AF4 fusion gene, andtheir sense strands are fully complementary to the antisense strandsexcept for the two 3′-terminal nucleotides on the antisense strand.However, it is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of an MLL-AF4mRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of Table 1, exceptthat not more than 1, 2 or 3 nucleotides per strand, respectively, havebeen substituted by other nucleotides (e.g. adenosine replaced byuracil), while essentially retaining the ability to inhibit MLL-AF4expression in cultured human SEM cells, as defined below. These agentswill therefore possess at least 15 nucleotides identical to one of thesequences of Table 1, but 1, 2 or 3 base mismatches with respect toeither the target MLL-AF4 mRNA sequence or between the sense andantisense strand are introduced. Mismatches to the target MLL-AF4 mRNAsequence, particularly in the antisense strand, are most tolerated inthe terminal regions and if present are preferably in a terminal regionor regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′terminus, most preferably within 6, 5, 4, or 3 nucleotides of the5′-terminus of the sense strand or the 3′-terminus of the antisensestrand. The sense strand need only be sufficiently complementary withthe antisense strand to maintain the overall double stranded characterof the molecule.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, an iRNA agent contains sense andantisense strands, preferably paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred siRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 to 4, or preferably 2 or 3 nucleotides, in length at each end. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′-endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. siRNA agents can resemble inlength and structure the natural Dicer processed products from longdsRNAs. Embodiments in which the two strands of the siRNA agent arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and preferably a 3′ overhang are also within the invention.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to downregulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell, e.g. an SEM cell, that expressesthe target gene, e.g., the MLL-AF4 fusion gene, either endogenously orbecause it has been transfected with a construct from which the MLL-AF4fusion gene can be expressed. The level of target gene expression priorto and following contact with the candidate iRNA agent can be compared,e.g., on an mRNA or protein level. If it is determined that the amountof RNA or protein expressed from the target gene is lower followingcontact with the iRNA agent, then it can be concluded that the iRNAagent downregulates target gene expression. The level of target MLL-AF4RNA or MLL-AF4 protein in the cell can be determined by any methoddesired. For example, the level of target RNA can be determined byNorthern blot analysis, reverse transcription coupled with polymerasechain reaction (RT-PCR), or RNAse protection assay. The level of proteincan be determined, for example, by Western blot analysis orimmuno-fluorescence.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g., a 2′-O-mathyl group. This further iRNAagen can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting MLL-AF4 geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse or rat). For example, the iRNAagent can be administered to an animal, e.g., an animal engineered toexpress the MLL-AF4 gene fusion, and the iRNA agent evaluated withrespect to its biodistribution, stability, and its ability to inhibitMLL-AF4 gene expression.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; orantigen particles for immunohistochemistry).

The iRNA agent can be evaluated with respect to its ability to downregulate MLL-AF4 gene expression. Levels of MLL-AF4 gene expression invivo can be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. TargetMLL-AF4 mRNA can be detected by any desired method, including but notlimited to RT-PCR, Northern blot, branched-DNA assay, or RNAaseprotection assay. Alternatively, or additionally, MLL-AF4 geneexpression can be monitored by performing Western blot analysis ontissue extracts treated with the iRNA agent.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., dsRNA agents, thatmediate RNAi to inhibit expression of MLL-AF4.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because the are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein. Modified RNA as used herein refers to a molecule inwhich one or more of the components of the nucleic acid, namely sugars,bases, and phosphate moieties, are different from that which occur innature, preferably different from that which occurs in the human body.While they are referred to as modified “RNAs,” they will of course,because of the modification, include molecules which are not RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. It may be desirable to modify one or both of the antisenseand sense strands of an iRNA agent. As nucleic acids are polymers ofsubunits or monomers, many of the modifications described below occur ata position which is repeated within a nucleic acid, e.g., a modificationof a base, or a phosphate moiety, or the non-linking O of a phosphatemoiety. In some cases the modification will occur at all of the subjectpositions in the nucleic acid but in many, and in fact in most, cases itwill not. By way of example, a modification may only occur at a 3′ or 5′terminal position, may only occur in a terminal region, e.g. at aposition on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. E.g., a phosphorothioatemodification at a non-linking O position may only occur at one or bothtermini, may only occur in a terminal regions, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand, or may occur in double strand and single strand regions,particularly at termini. Similarly, a modification may occur on thesense strand, antisense strand, or both. In some cases, the sense andantisense strand will have the same modifications or the same class ofmodifications, but in other cases the sense and antisense strand willhave different modifications, e.g., in some cases it may be desirable tomodify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNAagents is their stabilization towards degradation in biologicalenvironments and the improvement of pharmacological properties, e.g.pharmacodynamic properties, which are further discussed below. Othersuitable modifications to a sugar, base, or backbone of an iRNA agentare described in co-owned PCT Application No. PCT/US2004/01193, filedJan. 16, 2004. An iRNA agent can include a non-naturally occurring base,such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits andmonomers for oligonucleotide synthesis. Exemplary monomers are describedin co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10,2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

The sense and antisense sequences of an iRNA agent can be palindromic.Exemplary features of palindromic iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets MLL-AF4 fusion, can haveenhanced resistance to nucleases.

One way to increase resistance is to identify cleavage sites and modifysuch sites to inhibit cleavage, as described in co-owned U.S.Application No. 60/559,917, filed on May 4, 2004. For example, thedinucleotides 5′-UA-3′, 5′-UG-3′, 5′-CA-3′, 5′-UU-3′, or 5′-CC-3′ canserve as cleavage sites. In certain embodiments, all the pyrimidines ofan iRNA agent carry a 2′-modification, and the iRNA agent therefore hasenhanced resistance to endonucleases. Enhanced nuclease resistance canalso be achieved by modifying the 5′ nucleotide, resulting, for example,in at least one 5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide whereinthe uridine is a 2′-modified nucleotide; at least one5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; at least one 5′-cytidine-adenine-3′ (5′-CA-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; atleast one 5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide; or at least one5′-cytidine-cytidine-3′ (5′-CC-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, as described in co-owned U.S. ApplicationNo. 60/574,744, filed on May 27, 2004. The iRNA agent can include atleast 2, at least 3, at least 4 or at least 5 of such dinucleotides.

Preferably, the 2′-modified nucleotides include, for example, a2′-modified ribose unit, e.g., the 2′-hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE andaminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substitutents are 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Nucleolytic cleavage can also be inhibited by the introduction ofphosphate linker modifications, e.g., phosphorothioate linkages. Thus,preferred iRNA agents include nucleotide dimers enriched or pure for aparticular chiral form of a modified phosphate group containing aheteroatom at a nonbridging position normally occupied by oxygen. Theheteroatom can be S, Se, Nr₂, or Br₃. When the heteroatom is S, enrichedor chirally pure Sp linkage is preferred. Enriched means at least 70,80, 90, 95, or 99% of the preferred form. Modified phosphate linkagesare particularly efficient in inhibiting exonucleolytic cleavage whenintroduced near the 5′- or 3′-terminal positions, and preferably the5′-terminal positions, of an iRNA agent.

5′ conjugates can also inhibit 5′-3′ exonucleolytic cleavage. While notbeing bound by theory, a 5′ conjugate, such as naproxen or ibuprofen,may inhibit exonucleolytic cleavage by sterically blocking theexonuclease from binding to the 5′-end of oligonucleotide. Even smallalkyl chains, aryl groups, or heterocyclic conjugates or modified sugars(D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

Thus, an iRNA agent can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the iRNA agent as well, e.g., theability to interact with a protein, e.g., a transport protein, e.g.,serum albumin, or a member of the RISC, or the ability of the first andsecond sequences to form a duplex with one another or to form a duplexwith another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Some NRM modifications caninhibit hybridization so it is preferable to use them only in terminalregions, and preferable to not use them at the cleavage site or in thecleavage region of a sequence which targets a subject sequence or gene,particularly on the antisense strand. They can be used anywhere in asense strand, provided that sufficient hybridization between the twostrands of the ds iRNA agent is maintained. In some embodiments it isdesirable to put the NRM at the cleavage site or in the cleavage regionof a sense strand, as it can minimize off-target silencing.

In most cases, NRM modifications will be distributed differentlydepending on whether they are comprised on a sense or antisense strand.If on an antisense strand, modifications which interfere with or inhibitendonuclease cleavage should not be inserted in the region which issubject to RISC mediated cleavage, e.g., the cleavage site or thecleavage region (As described in Elbashir et al., 2001, Genes and Dev.15: 188, hereby incorporated by reference). Cleavage of the targetoccurs about in the middle of a 20 or 21 nt antisense strand, or about10 or 11 nucleotides upstream of the first nucleotide on the target mRNAwhich is complementary to the antisense strand. As used herein cleavagesite refers to the nucleotides on either side of the cleavage site, onthe target or on the iRNA agent strand which hybridizes to it. Cleavageregion means the nucleotides within 1, 2, or 3 nucleotides of thecleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sense or antisense strand.

Tethered Ligands

The properties of an iRNA agent, including its pharmacologicalproperties, can be influenced and tailored, for example, by theintroduction of ligands, e.g. tethered ligands.

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. The ligand or tethered ligand may bepresent on the ligand-conjugated monomer when the ligand-conjugatedmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the growing strand. For example, a monomerhaving, e.g., an amino-terminated tether, e.g., TAP-(CH₂)_(n)NH₂ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer subunitinto the strand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor ligand-conjugated monomer by coupling the electrophilicgroup of the ligand with the terminal nucleophilic group of theprecursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophilic moleculeses, lipids, lectins, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), proteins, protein bindingagents, integrin targeting molecules, polycationics, peptides,polyamines, and peptide mimics.

The ligand may be a naturally occurring or recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.Examples of polyamino acids include polylysine (PLL), poly L-asparticacid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic moieties, e.g., cationic lipid,cationic porphyrin, quaternary salt of a polyamine, or an alpha helicalpeptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a thyrotropin, melanotropin, surfactant proteinA, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin,bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGDpeptide mimetic.

Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. lowdensity lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA),or peptides, e.g., molecules having a specific affinity for a co-ligand,or antibodies e.g., an antibody, that binds to a specified cell typesuch as a cancer cell, endothelial cell, or bone cell. Ligands may alsoinclude hormones and hormone receptors. They can also includenon-peptidic species, such as cofactors, multivalent lactose,multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine,multivalent mannose, or multivalent fucose. The ligand can be, forexample, a lipopolysaccharide, an activator of p38 MAP kinase, or anactivator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., liver tissue, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient,which is taken up by a target cell, e.g., a proliferating cell. Theseare particularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include the B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells.

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

5′-Phosphate Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include aphosphoryl analog at the 5′ prime terminus. 5′-phosphate modificationsof the antisense strand include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

The sense strand can be modified in order to inactivate the sense strandand prevent formation of an active RISC, thereby potentially reducingoff-target effects. This can be accomplished by a modification whichprevents 5′-phosphorylation of the sense strand, e.g., by modificationwith a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATPrequirements and small interfering RNA structure in the RNA interferencepathway. Cell 107, 309-321.) Other modifications which preventphosphorylation can also be used, e.g., simply substituting the 5′-OH byH rather than O-Me. Alternatively, a large bulky group may be added tothe 5′-phosphate turning it into a phosphodiester linkage.

Transport of iRNA Agents into Cells

Not wishing to be bound by any theory, the chemical similarity betweencholesterol-conjugated iRNA agents and certain constituents oflipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) maylead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL)in blood and/or the interaction of the iRNA agent with cellularcomponents having an affinity for cholesterol, e.g. components of thecholesterol transport pathway. Lipoproteins as well as theirconstituents are taken up and processed by cells by various active andpassive transport mechanisms, for example, without limitation,endocytosis of LDL-receptor bound LDL, endocytosis of oxidized orotherwise modified LDLs through interaction with Scavenger receptor A,Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver,pinocytosis, or transport of cholesterol across membranes by ABC(ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 orABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoyfacilitated uptake by cells possessing such transport mechanisms, e.g.cells of the liver. As such, the present invention provides evidence andgeneral methods for targeting iRNA agents to cells expressing certaincell surface components, e.g. receptors, by conjugating a natural ligandfor such component (e.g. cholesterol) to the iRNA agent, or byconjugating a chemical moiety (e.g. cholesterol) to the iRNA agent whichassociates with or binds to a natural ligand for the component (e.g.LDL, HDL).

Other Embodiments

An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g.,from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc.Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of an iRNA agent and one that produces atranscript that includes the bottom strand of an iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto siRNA agent fragments that mediate gene silencing.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

An iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes two or more iRNAagents, e.g., a second iRNA agent that can mediate RNAi with respect toa second gene, or with respect to the same gene. Still otherpreparations can include at least three, five, ten, twenty, fifty, or ahundred or more different iRNA species. Such iRNAs can mediated RNAiwith respect to a similar number of different genes.

Where the two or more iRNA agents in such preparation target the samegene, they can have target sequences that are non-overlapping andnon-adjacent, or the target sequences may be overlapping or adjacent.

Optionally, the iRNA agents of the invention may be formulated usingliposomes. Liposomes of various compositions can be used forsite-specific delivery of various pharmaceutically active ingredients(Witschi, C. et al., Pharm. Res., 1999, 16:382-390; Yeh, M. K. et al.,Pharm. Res., 1996, 1693-1698). The interaction between the liposomes andthe cargo usually relies on hydrophobic interactions or chargeattractions, particularly in the case of cationic lipid delivery systems(Zelphati, O. et al., J. Biol. Chem., 2001, 276:35103-35110). HIV tatpeptide-bearing liposomes have also been constructed and used to delivercargo directly into the cytoplasm, bypassing the endocytotic pathway(Torchilin V. P. et al., Biochim. Biophys. Acta—Biomembranes, 2001,1511:397-411; Torchilin V. P. et al., Proc. Natl. Acad. Sci. USA, 2001,98:8786-8791). When encapsulated in sugar-grafted liposomes, pentamidineisethionate and a derivative have been found to be more potent incomparison to normal liposome-encapsulated drug or to the free drug(Banerjee, G. et al., J. Antimicrob. Chemother., 1996, 38(1):145-150). Athermo-sensitive liposomal taxol formulation (heat-mediated targeteddrug delivery) has been administered in vivo to tumor-bearing mice incombination with local hyperthermia, and a significant reduction intumor volume and an increase in survival time was observed compared tothe equivalent dose of free taxol with or without hyperthermia (Sharma,D. et al., Melanoma Res., 1998, 8(3):240-244). Topical application ofliposome preparations for delivery of insulin, IFN-alpha, IFN-gamma, andprostaglandin E1 have met with some success (Cevc G. et al., Biochim.Biophys, Acta, 1998, 1368:201-215; Foldvari M. et al., J. Liposome Res.,1997, 7:115-126; Short S. M. et al., Pharm. Res., 1996, 13:1020-1027;Foldvari M. et al., Urology, 1998, 52(5):838-843; U.S. Pat. No.5,853,755).

Antibodies represent another targeting device that may make liposomeuptake tissue-specific or cell-specific (Mastrobattista, E. et al.,Biochim. Biophys. Acta, 1999, 1419(2):353-363; Mastrobattista, E. etal., Adv. Drug Deliv. Rev., 1999, 40(1-2):103-127). The liposomeapproach offers several advantages, including the ability to slowlyrelease encapsulated active ingredients, the capability of evading theimmune system and proteolytic enzymes, and the ability to target tumorsand cause preferentially accumulation in tumor tissues and theirmetastases by extravasation through their leaky neovasculature. Othercarriers have also been used to deliver anti-cancer drugs to neoplasticcells, such as polyvinylpyrrolidone nanoparticles and maleylated bovineserum albumin (Sharma, D. et al., Oncol. Res., 1996, 8(7-8):281-286;Mukhopadhyay, A. et al., FEBS Lett., 1995, 376(1-2):95-98). Thus, usingtargeting and encapsulation technologies, which are very versatile andamenable to rational design and modification, delivery of an iRNA agentof the invention to desired cells can be facilitated.

As indicated above, the pharmaceutical composition of the presentinvention can include a liposome component. According to the presentinvention, a liposome comprises a lipid composition that is capable offusing with the plasma membrane of a cell, thereby allowing the liposometo deliver a cargo, e.g. a nucleic acid molecule composition, into acell. Some preferred liposomes include those liposomes commonly used ingene delivery methods known to those of skill in the art. Some preferredliposome delivery vehicles comprise multilamellar vesicle (MLV) lipidsand extruded lipids, although the invention is not limited to suchliposomes. Methods for preparation of MLVs are well known in the art.“Extruded lipids” are also contemplated. Extruded lipids are lipids thatare prepared similarly to MLV lipids, but which are subsequentlyextruded through filters of decreasing size, as described in Templetonet al., Nature Biotech., 1997, 15:647-652, which is incorporated hereinby reference in its entirety. Small unilamellar vesicle (SUV) lipids canalso be used in the compositions and methods of the present invention.Other preferred liposome delivery vehicles comprise liposomes having apolycationic lipid composition (i.e., cationic liposomes). For example,cationic liposome compositions include, but are not limited to, anycationic liposome complexed with cholesterol, and without limitation,include DOTMA and cholesterol, DOTAP and cholesterol, DOTIM andcholesterol, and DDAB and cholesterol. Liposomes utilized in the presentinvention can be any size, including from about 10 to 1000 nanometers(nm), or any size in between.

A liposome delivery vehicle can be modified to target a particular sitein a mammal, thereby targeting and making use of an iRNA agent of thepresent invention at that site. Suitable modifications includemanipulating the chemical formula of the lipid portion of the deliveryvehicle. Manipulating the chemical formula of the lipid portion of thedelivery vehicle can elicit the extracellular or intracellular targetingof the delivery vehicle. For example, a chemical can be added to thelipid formula of a liposome that alters the charge of the lipid bilayerof the liposome so that the liposome fuses with particular cells havingparticular charge characteristics. In one embodiment, other targetingmechanisms, such as targeting by addition of exogenous targetingmolecules to a liposome (i.e., antibodies) may not be a necessarycomponent of the liposome of the present invention, since effectiveimmune activation at immunologically active organs can already beprovided by the composition when the route of delivery is intravenous orintraperitoneal, without the aid of additional targeting mechanisms.However, in some embodiments, a liposome can be directed to a particulartarget cell or tissue by using a targeting agent, such as an antibody,soluble receptor or ligand, incorporated with the liposome, to target aparticular cell or tissue to which the targeting molecule can bind.Targeting liposomes are described, for example, in Ho et al.,Biochemistry, 1986, 25: 5500-6; Ho et al., J Biol Chem, 1987a, 262:13979-84; Ho et al., J Biol Chem, 1987b, 262: 13973-8; and U.S. Pat. No.4,957,735 to Huang et al., each of which is incorporated herein byreference in its entirety). In one embodiment, if avoidance of theefficient uptake of injected liposomes by reticuloendothelial systemcells due to opsonization of liposomes by plasma proteins or otherfactors is desired, hydrophilic lipids, such as gangliosides (Allen etal., FEBS Lett, 1987, 223: 42-6) or polyethylene glycol (PEG)-derivedlipids (Klibanov et al., FEBS Lett, 1990, 268: 235-7), can beincorporated into the bilayer of a conventional liposome to form theso-called sterically-stabilized or “stealth” liposomes (Woodle et al.,Biochim Biophys Acta, 1992, 1113: 171-99). Variations of such liposomesare described, for example, in U.S. Pat. No. 5,705,187 to Unger et al.,U.S. Pat. No. 5,820,873 to Choi et al., U.S. Pat. No. 5,817,856 toTirosh et al.; U.S. Pat. No. 5,686,101 to Tagawa et al.; U.S. Pat. No.5,043,164 to Huang et al., and U.S. Pat. No. 5,013,556 to Woodle et al.,all of which are incorporated herein by reference in their entireties).

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent, e.g., an iRNA agent thattargets MLL-AF4, can be delivered to a subject by a variety of routes.Exemplary routes include intrathecal, parenchymal, intravenous, nasal,oral, and ocular delivery. The preferred means of administering the iRNAagents of the present invention is through parenteral administration.

An iRNA agent can be incorporated into pharmaceutical compositionssuitable for administration. For example, compositions can include oneor more species of an iRNA agent and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, or intrathecal or intraventricular administration.

The route of delivery can be dependent on the disorder of the patient.In general, the delivery of the iRNA agents of the present invention isdone to achieve systemic delivery into the subject. The preferred meansof achieving this is through parental administration.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

Administration can be provided by the subject or by another person,e.g., a another caregiver. A caregiver can be any entity involved withproviding care to the human: for example, a hospital, hospice, doctor'soffice, outpatient clinic; a healthcare worker such as a doctor, nurse,or other practitioner; or a spouse or guardian, such as a parent. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The term “co-administration” refers to administering to a subject two ormore agents, and in particular two or more iRNA agents. The agents canbe contained in a single pharmaceutical composition and be administeredat the same time, or the agents can be contained in separate formulationand administered serially to a subject. So long as the two agents can bedetected in the subject at the same time, the two agents are said to beco-administered.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Dosage. An iRNA agent can be administered at a unit dose less than about75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20,10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4×1016copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmole of RNA agent per kg of bodyweight. The unit dose, for example, canbe administered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into an organ), an inhaled dose, or a topicalapplication.

Delivery of an iRNA agent directly to an organ (e.g., directly to theliver) can be at a dosage on the order of about 0.00001 mg to about 3 mgper organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

The dosage can be an amount effective to treat or prevent a disease ordisorder.

In one embodiment, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time. Because iRNA agent mediated silencing can persist forseveral days after administering the iRNA agent composition, in manyinstances, it is possible to administer the composition with a frequencyof less than once per day, or, for some instances, only once for theentire therapeutic regimen.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70,60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or0.0005 mg per kg of bodyweight per day. The maintenance doses arepreferably administered no more than once every 5, 10, or 30 days.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease, its severityand the overall condition of the patient. In preferred embodiments thedosage may be delivered no more than once per day, e.g., no more thanonce per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8days. Following treatment, the patient can be monitored for changes inhis condition and for alleviation of the symptoms of the disease state.The dosage of the compound may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound featured in the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. It will also be appreciated thatthe effective dosage of an iRNA agent such as an siRNA agent used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein. For example, thesubject can be monitored after administering an iRNA agent composition.Based on information from the monitoring, an additional amount of theiRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Example 2 siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Glen Research, Sterling Va.) as solid support. RNA and RNAcontaining 2′-O-methyl nucleotides were generated by solid phasesynthesis employing the corresponding phosphoramidites and 2′-O-methylphosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,Germany). These building blocks were incorporated at selected siteswithin the sequence of the oligoribonucleotide chain using standardnucleoside phosphoramidite chemistry such as described in Currentprotocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.),John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkageswere introduced by replacement of the iodine oxidizer solution with asolution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification by anion exchange HPLC of the crudeoligoribonucleotides were carried out according to establishedprocedures. Yields and concentrations were determined by UV absorptionof a solution of the respective RNA at a wavelength of 260 nm using aspectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim,Germany). Double stranded RNA was generated by mixing an equimolarsolution of complementary strands in annealing buffer (20 mM sodiumphosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at85-90° C. for 3 minutes and cooled to room temperature over a period of3-4 hours. The purified RNA solution was stored at −20° C. until use.

Example 3 siRNA Design

In order to identify efficient MLL-AF4 siRNAs, we performed an siRNAscan of the sequence including and flanking either side of the MLL-AF4fusion site (Domer et al., Proc Natl Acad Sci USA 90:7884-7888, 1993; Guet al., Cell 71:701-708, 1992; Marshalek et al., Br. J. Haematology90:308-320, 1995). To perform the scan, we synthesized 14 differentsiRNAs having target sites that were overlapping and moved step-wise byone nucleotide down the sequence including the MLL-AF4 fusion site. ThesiRNA sequences and their target sites on the fusion mRNA are shown inTable 2. The siRNA duplexes shown in Table 2 are referred to hereinbelowas siMAX, wherein X stands for the integer number in the denomination ofthe sense and antisense strand making up the respective duplex in Table2. E.g., siMAS stands for the duplex formed from MASS and MASas.However, siMAx stands for the duplex formed by MA3s and MAxas (Agent No.2 in Table 1) and siMAmm for the mismatch duplex formed by MAmms andMAmmas.

In addition, one siRNA was synthesized homologous to the fusion sitevariant in RS4;11 cells (herein, siMARS). Position 1 to 21 of the sensestrand of siMARS correspond to Position 4338-4358 of Genbank accessionno. L22179 (version no. L22179.1 GI:347376; Domer, P. H., et al.; Proc.Natl. Acad. Sci. U.S.A. 1993; 90:7884-7888).

TABLE 2 MLL-AF4 siRNA sequences^(d).fusion mRNA: ACAAAAACCAAAAGAAAAG/CAGACCUACUCCAAUGAAGc (SEQ ID NO: 42)^(c)SEQ ID NO: MA1s^(a) 5′-AAG/CAGACCUACUCCAAUGAA-3′ 1 MA1as^(b)3′-UUUUC/GUCUGGAUGAGGUUACUU-5′ 2 MA2s 5′-AAAG/CAGACCUACUCCAAUGA-3′ 3MA2as 3′-CUUUUC/GUCUGGAUGAGGUUACU-5′ 4 MA3s 5′-AAAAG/CAGACCUACUCCAAUG-3′5 MA3as 3′-UCUUUUC/GUCUGGAUGAGGUUAC-5′ 6 MAxas3′-UCUUUUC/GUCUGGAUGAGGUU-5′ 7 MAmms 5′-AAAAG/CUGACCUUCUCCAAUG-3′ 8MAmmas 3′-UCUUUUC/GACUGGAAGAGGUUAC-5′ 9 MA4s5′-GAAAAG/CAGACCUACUCCAAU-3′ 10 MA4as 3′-UUCUUUUC/GUCUGGAUGAGGUUA-5′ 11MA5S 5′-AGAAAAG/CAGACCUACUCCAA-3′ 12 MA5as3′-UUUCUUUUC/GUCUGGAUGAGGUU-5′ 13 MA6s 5′-AAGAAAAG/CAGACCUACUCCA-3′ 14MA6as 3′-UUUUCUUUUC/GUCUGGAUGAGGU-5′ 15 MA7s5′-AAAGAAAAG/CAGACCUACUCC-3′ 16 MA7as 3′-GUUUUCUUUUC/GUCUGGAUGAGG-5′ 17MA8s 5′-AAAAGAAAAG/CAGACCUACUC-3′ 18 MA8as3′-GGUUUUCUUUUC/GUCUGGAUGAG-5′ 19 MA9s 5′-CAAAAGAAAAG/CAGACCUACU-3′ 20MA9as 3′-UGGUUUUCUUUUC/GUCUGGAUGA-5′ 21 MA10s5′-CCAAAAGAAAAG/CAGACCUAC-3′ 22 MA10as 3′-UUGGUUUUCUUUUC/GUCUGGAUG-5′ 23MA11s 5′-ACCAAAAGAAAAG/CAGACCUA-3′ 24 MA11as3′-UUUGGUUUUCUUUUC/GUCUGGAU-5′ 25 MA12s 5′-AACCAAAAGAAAAG/CAGACCU-3′ 26MA12as 3′-UUUUGGUUUUCUUUUC/GUCUGGA-5′ 27 MA13s5′-AAACCAAAAGAAAAG/CAGACC-3′ 28 MA13as 3′-UUUUUGGUUUUCUUUUC/GUCUGG-5′ 29MA14s 5′-AAAACCAAAAGAAAAG/CAGAC-3′ 30 MA14as3′-GUUUUUGGUUUUCUUUUC/GUCUG-5′ 31 siMARSs 5′-ACUUUAAGCAGACCUACUCCA-3′ 32siMARSas 3′-CCUGAAAUUCGUCUGGAUGAGGU-5′ 33 siAGF1s5′-CCUCGAAAUCGUACUGAGAAG-3′ 34 siAGF1as 3′-UUGGAGCUUUAGCAUGACUCU-5′ 35siAGF6s 5′-CCUCGAAUUCGUUCUGAGAAG-3′ 36 siAGF6as3′-UUGGAGCUUAAGCAAGACUCU-5′ 37 siGL2s 5′-CGUACGCGGAAUACUUCGATT-3′ 38siGL2as 3′-TTGCAUGCGCCUUAUGAAGCU-5′ 39 siK4s 5′-GAUGAGGAUCGUUUCGCAUGA-3′40 siK4as 3′-UCCUACUCCUAGCAAAGCGUACU-5′ 41 ^(a)“s”indicates sensestrand; ^(b)“as”indicates antisense strand; ^(c)“/”indicates the fusionsite between chromosome 11 and chromosome 4 sequence; ^(d)duplexed siRNAagents are referenced in the specification as “siMAn”, n = 1 to 14,siMAx for the duplex of MA3s with MAxas, and siMAmm for the duplexMAmms + MAmmas

As controls, the mismatch siRNA siMAmm, the siRNAs siAGF1 and itsmismatch control siAF6, targeting AML/MTG8, as well as siRNAs siK4 andsiGL2, targeting neomycin phosphotransferase mRNA (Heidenreich et al.,Blood 101:3157-3163, 2003) were used. Positions 1 to 21 in the sensestrand of siAGF1 correspond to positions 2102-2122 of AML1/MTG8, Genbankaccession no. D13979. siAGF6 is identical to siAGF1 except for A→Uswitches in positions 8 and 13 of the sense strand sequence, and thecorresponding changes to the antisense sequence. Positions 1 to 21 inthe sense strand of siGL2 correspond to positions 514-533 of P. pyralisluciferase, Genbank accession no. M15077. Positions 1 to 21 in the sensestrand of siK4 correspond to positions 4184-4202 of Neomycinphosphotransferase II, Genbank accession No. L11017.

The siRNAs, except siMAx, siAGF1, siAGF6, and siGL2 consisted of 21nucleotide-long sense and 23 nucleotide-long antisense strands, and thesiRNAs formed a single 3′-overhang of 2 nucleotides by the antisensestrand. siMAx, siAGF1, siAGF6, and siGL2 consisted of 21 nucleotide-longsense and antisense strands, forming 3′-overhangs of 2 nucleotides onboth ends.

Example 4 Cell Culture

The efficiencies of the different siRNAs to reduce the level of mRNAexpressed from the several genes studied was examined in cell lines SEM(Greil et al., Br J Haematol. 86:275-283, 1994), RS4;11 (Stong, R. C.,et al.; Blood 1985, 65:21-31) (obtained from the DSMZ, Braunschweig,Germany) and MV4;11 (Lange, P. H., and Winfield, H. N.; Cancer 1987;60:464-472) (obtained from J. Krauter, Medical School Hannover, Germany)which carry the chromosomal translocation t(4;11)(q21;q23), but expressdifferent MLL-AF4 variants due to different break points. Furtherleukaemic cell lines used in this study were HL60 (Collins, S. J., etal.; Nature 1977; 270:347-349), K562 (Lozzio, C. B., and Lozzio, B. B.;J Natl Cancer Inst 1973; 50:535-538.), Kasumi-1 (Asou, H., et al.; Blood1991; 77:2031-2036), SKNO-1 (Matozaki, S., et al.; Br J Haematol 1995;89:805-811) and U937 (Sundstrom, C., and Nilsson, K.; Int J Cancer 1976;17:565-577.). SKNO-1 cells were maintained in RPMI 1640 Glutamax medium(Invitrogen, Karlsruhe, Germany) supplemented with 20% FCS (SeraPlus,PAN Biotech GmbH) and 7 ng/ml GM-CSF, all other lines in RPMI 1640Glutamax medium supplemented with 10% FCS at 37° C. and 5% CO2. Primaryhuman CD34+ cells from bone marrow of healthy patients were obtained asfrozen stocks from University Children's Hospital in Tübingen.

Example 5 siRNA Treatment

SiRNA electroporations of SEM were carried out as described previously(Dunne et al., Oligonucleotides, 13:375-80, 2003; and Heidenreich, etal., Blood 101:3157-3163, 2003). Electroporation was performed with aFischer electroporator (Fischer, Heidelberg, Germany) using a rectanglepulse of 350 V for 10 ms. After incubation for 15 minutes at roomtemperature, the cells were diluted twenty fold with culture medium andincubated at 37° C. and 5% CO₂.

Example 6 RT-PCR

Levels of endogenous MLL-AF4, MLL, AF4, HOXA7, HOXA9, OAS1 and STAT1were assayed by Real-Time PCR. Total RNA extraction was performed withthe Rneasy kit (Qiagen, Hilden, Germany) as suggested by themanufacturer. One mg of total RNA was subsequently used for thereal-time reverse transcription coupled to polymerase chain reaction(RT-PCR). Reverse transcription reactions were performed using 25 mMrandom hexamers, 5×RT buffer, 1 mM dNTP Mix, 20 U RNase inhibitor (MBI)and 100 U MMLV-RT, RNase H-(Promega, Heidelberg, Germany). The mixturewas incubated at room temperature for 10 min, for 45 min at 42° C., and3 min at 99° C. mRNA levels of the respective genes were normalized toGAPDH mRNA levels.

Real-time PCR reaction was performed using primers for MLL-AF4, MLL,AF4, HOXA7, HOXA9, OAS1 and STAT1. Primers hybridizing to GAPDH wereused as a control. The master mix (MLL-AF4) for the TaqMan real-time PCRreaction contained 62.5 nM of forward and reverse primers, 62.5% (v/v)Sybr-Green Mix. The master mix for the GAPDH reaction contained 375 nMforward and reverse primers and 62.5% (v/v) Sybr-Green-Mix. Otherwise,RT-PCR was performed as described in Martinez et al., BMC Cancer 2004,4:44. The sequences of the primers used are listed in Table 3. Primerswere designed with PRIMER-EXPRESS software (Applied Biosystems, FosterCity, Calif., USA).

TABLE 3 PCR primer sequences SEQ ID Gene Sense/antisense primer NO:MLL-AF4 5′-ACAGAAAAAAGTGGCTCCCCG-3′/ 43 5′-TATTGCTGTCAAAGGAGGCGG-3′ 44MLL: 5′-ACAGAAAAAAGTGGCTCCCCG-3′/ 45 5′-GCAAACCACCCTGGGTGTTA-3′ 46 AF4:5′-CAGAAGCCCACGGCTTATGT-3′/ 47 5′-TATTGCTGTCAAAGGAGGCGG-3′ 48 HOXA7:5′-CGCCAGACCTACACGCG-3′/ 49 5′-CAGGTAGCGGTTGAAGTGGAA-3′ 50 HOXA9:5′-CCACCATCCCCGCACA-3′/ 51 5′-AACAGGGTTTGCCTTGGAAA-3′ 52 OAS1:5′-TCCAAGGTGGTAAAGGGTGG-3′/ 53 5′-AGGTCAGCGTCAGATCGGC-3′ 54 GAPDH:5′-GAAGGTGAAGGTCGGAGTC-3′/ 55 5′-GAAGATGGTGATGGGATTTC-3′ 56 STAT1:5′-CATCACATTCACATGGGTGGA-3′/ 57 5′-GGTTCAACCGCATGGAAGTC-3′ 58

Example 7 Colony Formation Assay

Twenty-four hours after cell electroporation with siRNAs, 10,000 cellswere plated in 0.5 ml of RPMI 1640 medium containing 20% FCS and 0.56%methylcellulose, in 24-well plates. In the case of RS4;11, cell numberswere increased to 20,000 per well. Colonies containing more than 20cells were counted 14 days after plating. Under these conditions,mock-transfected cells (electroporated without siRNAs) yielded 50 to 100colonies per well dependent on the cell line examined. HumanColony-Forming Cell Assays were performed using MethoCultMethylcellulose-based media (CellSystems, St. Katharinen, Germany).After electroporation, 5000 human primary CD34+ cells were plated induplicate in 35-mm culture dishes with 1 ml of methylcellulose medium.The number of CFU-GEMMs and CFU-GMs was counted 10 days after theplating.

Example 8 MTT Test

Cells were electroporated twice within 48 h and were plated on 96-wellplates at the density of 0.5×105 cells in 100 μl/well. Every 24 h later,10 μl of MTT solution (Roche, Mannheim, Germany) was added. Afterincubation for 4 hours at 37° C., cells were lysed with thesolubilization solution according to the manufacturer's instruction. TheOD measurements were performed using ELISA Reader (Dynex, Frankfurt/M,Germany) at 560 nm, and 650 nm as a reference wavelength. Cell numberswere calculated by cell dilution series. Human Colony-Forming CellAssays were performed using MethoCult® Methylcellulose-based media(CellSystems, St. Katharinen, Germany). The human primary cells wereelectroporated and 5000 cells were subsequently plated in 35-mm culturedishes with 1 ml of methylcellulose medium. Each sample was plated induplicate and the number of CFU-GEMMs and CFU-GMs was counted 10 daysafter the plating.

Example 9 Cell Cycle Analysis and Apoptosis Assay

Cell cycle analysis was performed as described previously (32). Theobtained data were subsequently analyzed and evaluated using ModFitprogram (Verity, Topsham, USA). Apoptosis was examined with humanAnnexin V/FITC Kit (Bender MedSystems, Wien, Austria) according to theprovider's instructions. Briefly, 2−5×105 cells were washed with PBS atthe indicated time points after electroporation followed by incubationin the presence of Annexin-V-FITC solution for 10 min at roomtemperature. The cells were washed again with PBS and stained withpropidium iodide. The samples were then immediately analyzed by flowcytometry using a FACSCalibur (Becton Dickinson, Heidelberg, Germany).

Example 10 Western Blotting

To obtain total cellular protein, proteins present in the flow-throughof RNeasy columns were precipitated with two volumes of acetone anddissolved in urea buffer (9 M urea, 4% (w/w)3-[(3-Cholamidopropyl)-dimethylammonio]-propansulfonat (CHAPS), 1% (w/w)Dithiothreitol). Total lysates (50 μg for MLL-AF4 detection, 10 μg forall other immunoblots) were analyzed as described (32). The followingantibodies were used for immunoblot detection: Cleaved caspase-3(Asp175) (1:1000, Cell Signaling Technology, #9661); Tubulin Ab-4 (1mg/l, NeoMarkers MS-719-P0, Fremont, USA) Bcl-XL (1:500, BD PharMingen,#556499); MLLT2 (1:600, Orbigen, #10852); GAPDH (1:20,000, HyTest,#5G4).

Example 11 Xenotransplantation of SCID Mice

Female 4-5 week old CB-17/lcrCrj-SCID/SCID mice were obtained fromCharles River Germany. SEM cells (2×107) were electroporated on day oneand day 3 either without (Mock) or with 500 nM of the indicated siRNA.On day 4, cells were intraperitoneally injected into mice. Animals weremaintained and treated according to protocols approved by the RegionalBoard Tübingen.

Example 12 Histology

Organs were removed and fixed in neutrally buffered 4% formalin at roomtemperature for 4-5 days followed by dehydration, embedding intoparaffin and sectioning. The tissues were stained with hematoxylin(Meyer's hemalum solution, Merck, Darmstadt, Germany) and eosin (EosinY, Merck, Darmstadt, Germany) for light microscopy. Light microscopy wasperformed with a Zeiss Axioplan microscope (Zeiss, Guttingen, Germany)using a 20× Plan-Neofluar or 40× Plan-Neofluar 1.3 oil lens. Images werecaptured using Axio Vision 4 Software provided with the microscope andAdobe Photoshop (Adobe Systems, San Jose, Calif., USA).

Example 13 Statistical Analyses

Colony formation assays were analyzed by unpaired student's t-test.Survival curves were analyzed by log-rank test.

Example 14 Identification of siRNAs with Activity Towards Inhibition ofMLL-AF4 Expression

To identify highly efficient MLL-AF4 siRNAs, we performed an siRNA scanof the MLL-AF4 fusion site. For that, we synthesized 14 different siRNAswith target sites moved by one single nucleotide each. The efficienciesof the different siRNAs were examined in the t(4;11)-positive leukemiccell line SEM established from a 5 year old ALL patient in relapse(Greil, J., et al.; Br J Haematol 1994; 86:275-283). Of all 14 siRNAsexamined, two siRNAs, siMA3 and siMA6, diminished MLL-AF4 mRNA levels bymore than 60% (FIG. 1A). The reduction of MLL-AF4 transcript levels wasdose-dependent and reached its maximum of 70% with 750 nM siRNA (datanot shown). Time course experiments showed that MLL-AF4 mRNA amountsreached their minimum between 24 to 48 hours after siRNA transfectionand recovered to normal levels at day 4 (FIG. 1B). Moreover, siMA6affected neither wildtype AF4 nor MLL mRNA levels (FIG. 1C), whereassiMA3 substantially reduced AF4 levels (data not shown). The mismatchcontrol siRNA siMM affected neither MLL-AF4 nor the correspondingwildtype allele transcripts. The decrease in MLL-AF4 mRNA levels wasreflected by a concomitant 67% decrease in MLL-AF4 protein (FIG. 1D).

The MLL-AF4 fusion site varies between different t(4;11) positive celllines. Whereas SEM cells express a transcript containing an MLL exon 9AF4 exon 4 (e9-e4) fusion, RS4;11 express an exon 10-exon 4 (e10-e4)variant. Inspite of a homology of 67%, siMA6 did not diminish levels ofthe e10-e4 isoform in RS4;11 cells (Domer, P. H., et al.; Proc Natl AcadSci USA 1993; 90:7884-7888), whereas a perfectly homologous siRNA,siMARS, reduced MLL-AF4 e10-e4 in RS4;11 by 60%, without affecting AF4or MLL expression.

Neither siMA6 nor siMM induced STAT1 or 2′-5′-oligoadenylate synthase 1expression (FIG. 1F) indicating that these siRNAs did not trigger aninterferon response (Sledz, C. A., et al.; Nat Cell Biol 2003;5:834-839). Transfection with polyIC increased OAS1 transcript levelsmore than fiftyfold and STAT1 mRNA levels more than tenfold (FIG. 1F)demonstrating the inducibility of interferon response pathways in theseleukemic cells. Because of their high specificity, the MLL-AF4 siRNAsiMA6 and the mismatch control siRNA siMM were chosen to proof thesignificance of MLL-AF4 expression for the leukemic phenotype.

Example 15 MLL-AF4 Affects Leukemic Clonogenicity

To study the relevance of MLL-AF4 for leukemic clonogenicity, wetransfected t(4;11)-positive SEM cells with siRNAs followed byincubation in semisolid medium. SiMA6-mediated depletion of MLL-AF4reduced the number of colonies fivefold (FIG. 2A). This effect wasspecific, since colony formation of the t(8;21)-positive leukemic cellline Kasumi-1 was not affected by siMA6. Vice versa, transfection withthe AML1/MTG8-specific siRNA siAGF1 compromised Kasumi-1 colonyformation without interfering with SEM colony formation (Martinez, N.,et al.; BMC Cancer 2004; 4:44). None of the mismatch controls (siMM andsiAGF6) affected leukemic clonogenicity. Furthermore, neither of thet(4;11)-negative leukemic cell lines HL60, K562, SKNO-1 and U937 nor thet(4;11)-positive cell lines RS4;11 and MV4;11 expressing MLL-AF4variants not affected by siMA6 showed impaired colony formation uponsiRNA transfection (data not shown). Notably, siMARS-mediatedsuppression of the MLL-AF4 e10-e4 variant reduced RS4;11 colonyformation twofold, demonstrating the dependence of clonogenic efficacyon MLL-AF4 for another t(4;11) cell line (FIG. 2B). MLL-AF4 siRNAelectroporation of primary human hemopoetic CD34+ cells did neitheraffect the numbers of GEMM nor GM colonies (FIG. 2C). This lack ofeffect cannot be attributed to inefficient siRNA transfections, sinceboth the cell lines used here and the human hemopoetic CD34+ cells thecan be efficiently transfected with functional siRNAs (Scherr, M., etal.; Blood 2003; 101:1566-1569; Heidenreich, O., et al.; Blood 2003;101:3157-3163; Dunne, J., et al.; Oligonucleotides 2003; 13:375-380.).

Example 16 Suppression of MLL-AF4 Inhibits Leukemic Proliferation andCell Cycle Progression

Next, we examined the role of MLL-AF4 in the control of leukemicproliferation in suspension culture. Whereas a single electroporationwith siMA6 did not affect the doubling time of t(4;11)-positive SEMcells (data not shown), repeating siRNA electroporations for everysecond day resulted in a sustained inhibition of proliferation of SEMcells by siMA6, and of RS4;11 cells by siMARS (FIG. 2D). Thus,proliferation was only inhibited by the siRNA homologous to thecorresponding MLL-AF4 fusion site demonstrating the specificity of theseMLL-AF4 siRNAs. Mock or siRNA-electroporated SEM or RS4;11 cellsproliferated with a doubling time of 1.4 days demonstrating that therepeated electroporation did not seriously affect their proliferation.

The reduced proliferation of t(4;11)-positive cells upon MLL-AF4depletion was paralleled by changes in the cell cycle distribution.During a time course of 10 days with repetitive MLL-AF4 siRNAelectroporations, the fraction of S phase cells decreased in both SEMand RS4;11 cells from 50% to 30% and 20%, respectively, with aconcomitant increase in the fraction of G0/G1 phase cells (FIG. 2E).Notably, siMA6 affected cell cycle distribution only in SEM cells,whereas siMARS caused those changes only in RS4;11 cells. Thus,depletion of MLL-AF4 negatively interferes with the progression oft(4;11)-positive cells from G1 to S phase. The impaired G1/S transitionis not associated with cellular senescence, as senescence-associatedβ-galactosidase activity did not increase upon MLL-AF4 depletion (datanot shown).

Example 17 MLL-AF4 Depletion Induces Apoptosis in t(4;11)-Positive SEMCells

Cell cycle analysis of SEM and RS4;11 cells revealed that the continuousdepletion of MLL-AF4 for ten days raised the number of subG1 cellstenfold compared to controls, indicating an increased amount ofapoptotic cells (FIG. 3A). Staining with annexin V and propidium iodidealso demonstrated for SEM cells a threefold increase in apoptotic cellsupon suppression of MLL-AF4 for four days thereby agreeing with theincrease in sub G1 cells seen at the same time point (FIG. 3B). Notably,the almost inactive siRNA siMA13 (see FIG. 1A) only marginally affectedthe amount of apoptotic cells suggesting a direct correlation betweenthe extent of MLL-AF depletion and the rate of apoptosis. This resultwas corroborated by the proteolytic activation of caspase-3 anddecreased amounts of the anti-apoptotic protein Bcl-XL upon transfectionwith siMA6 (FIG. 3C).

Example 18 MLL-AF4 Suppression Decreases Expression of Hox Genes

Since expression of MLL oncoproteins including MLL-AF4 is associatedwith increased expression of several Hox genes including Hoxa7 and Hoxa9as well as of another homeotic gene, Meis1. Therefore, we analyzed theexpression of these three genes in dependence on the MLL-AF4 level.After two consecutive transfections of SEM cells with the MLL-AF4 siRNAsiMA6, Hoxa7 mRNA levels decreased twofold (FIG. 4). A minor reductionof Hoxa9 was also observed. In RS4;11 cells, Hoxa7, but not Hoxa9 wasreduced upon transfection with siMARS. The absence of Hoxa9 reduction inRS4;11 cells might be due to the lower efficiency of the MLL-AF4suppression compared to that in SEM cells (FIG. 1D, E).

Example 19 MLL-AF4 is Important for the Leukemic Engraftment oft(4;11)-Positive Cells

Leukemic cell growth in SCID mice has been shown to be associated withhigh-risk B-ALL (Uckun, F. M., et al.; Blood 1995; 85:873-878).Therefore, we used an t(4;11)-SCID mouse model to ask, whethersiRNA-mediated depletion of MLL-AF4 affects engraftment and thedevelopment of leukemic disease in vivo (Pocock, C. F., et al.; Br JHaematol 1995; 90:855-867). Intraperitoneal transplantation of eithermock- or control siRNA-treated SEM cells into SCID mice resulted in a100% leukemia-associated mortality within 70 days post transplantationwith a median survival of 52 and 52 days, respectively (FIG. 5A).Xenotransplantation of MLL-AF4-depleted SEM cells caused a significantlyless severe phenotype with a median survival of 82 days and an overallsurvival of 38% at day 125 (p<0.01). Animals succumbing the diseaseshowed ovarian tumors, splenomegaly and massive leukemic blastinfiltration in bone marrow, spleen and liver, whereas the organs of asurviving animal of the siMA6 group showed no signs of leukemicinfiltration 228 days after transplantation (FIG. 5B, C). In conclusion,siRNA-mediated suppression of MLL-AF4 reduced the leukemic engraftmentof t(4;11)-positive cells in xenotransplanted SCID mice.

Example 20 Conclusions

Inhibition of MLL-AF expression diminished leukemic proliferation insuspension culture as well as colony formation of t(4;11) cell lines.This reduced clonogenicity and proliferation was accompanied by anincrease in apoptosis. Furthermore, depletion of MLL-AF4 was accompaniedby a decrease in Hoxa7, Hoxa9 and Meis-1 expression, which in turn maycontribute to apoptosis. Finally, siRNA-mediated MLL-AF4 suppressionseriously compromised the leukemic engraftment in xenotransplantatedSCID mice. Since efficient engraftment in SCID mice predicts anincreased probability of relapse in ALL patients (Uckun, F. M., et al.;Blood 1995; 85:873-878), these data suggest that interfering withMLL-AF4 functions may improve patients outcome.

Because of its exclusive expression in t(4;11) leukemic cells, andbecause of its central role in the maintenance of leukemia, MLL-AF4 is avery promising target for the treatment of this highly aggressiveleukemia. However, currently no small molecule inhibitors specific forthis fusion protein are available. We show that siRNAs homologous to thefusion site efficiently suppress MLL-AF4. Moreover, we demonstrate thattwo different variants of this fusion gene can be targeted with highefficacy and exclusive specificity. This exclusive specificity alsoproves that the observed antileukemic properties of these siRNAs aredirectly due to MLL-AF4 suppression and not to off-target effects, suchas unintended inhibition of other genes, or induction of interferonresponse. Our results suggest that MLL-AF4 siRNAs may provide aspecific, but still flexible and, thus, promising therapeutic tool forthe treatment of t(4;11) ALL

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

1. A method for treating a disorder associated with MLL-AF4 fusionexpression comprising administering to a subject having or at risk fordeveloping the disorder a composition comprising a double stranded iRNAagent, wherein the iRNA agent comprises a sense strand and an antisensestrand each 15-30 nucleotides in length and a double stranded region15-30 basepairs in length, wherein the antisense strand comprises aregion of complementarity to a mRNA encoding an MLL-AF4 fusion gene,wherein the region of complementarity is less than 30 nucleotides inlength, and the antisense strand comprises at least 15 contiguousnucleotides from an antisense sequence of agents 1-12 provided in Table1 (SEQ ID NOs 6, 7, 11, 13, 15, 19, 21, 23, 25, 27, 31, and 33).
 2. Themethod of claim 1, wherein the iRNA agent is selected from the groupconsisting of agents 1-12 provided in Table
 1. 3. The method of claim 1,wherein the disorder is a proliferative disorder, cancer, leukemia, oracute lymphoblastic leukemia and/or is characterized by the presence ofa t(4;11) chromosomal translocation and/or the presence of an MLL-AF4fusion gene.
 4. The method of claim 1, wherein the subject is human. 5.The method of claim 1, wherein the iRNA agent comprises at least onenucleotide modification.
 6. The method of claim 5, wherein thenucleotide modification is a phosphorothioate or a 2′ modifiednucleotide.
 7. The method of claim 5, wherein the nucleotidemodification is a 2′ sugar modification, a modification in a singlestrand overhang, a 5′-modification which includes one or more phosphategroups or one or more analogs of phosphate groups.
 8. The method ofclaim 5, wherein the nucleotide modification is a 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide;a 5′-uridineguanine-3′ (5′-UG-3′) dinucleotide, wherein the 5′-uridineis a 2′-modified nucleotide; a 5′-cytidine-adenine-3′ (5′-CA-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; or a5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide.
 9. The method of claim 5, wherein the2′-modification is chosen from the group consisting of: 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
 10. The method ofclaim 1, wherein the iRNA agent comprises a cholesterol moiety.
 11. Themethod of claim 10, wherein the cholesterol moiety is conjugated to the3′-end of the sense strand of the iRNA agent.
 12. The method of claim 1,wherein the iRNA agent comprises at least one nucleotide overhang having1 to 4 unpaired nucleotides.
 13. The method of claim 12, wherein thenucleotide overhang is a two nucleotide overhang at the 3′ end of theantisense strand.
 14. The method of claim 1, wherein the compositioncomprises a pharmaceutically acceptable carrier.
 15. The method of claim14, wherein the pharmaceutically acceptable carrier comprises apolycation or a lipid or a lipid based molecule.
 16. The method of claim1, wherein the composition comprises a ligand.
 17. The method of claim16, wherein the ligand is a lipid or lipid-based molecule.
 18. Themethod of claim 1, wherein the method (a) reduces MLL-AF4 fusion geneexpression in a cell or tissue of the subject by at least 2%; (b) doesnot substantially reduce wildtype MLL and/or AF4 mRNA levels of thesubject; (c) does not trigger an interferon response; (d) inhibitsleukemic proliferation; (e) is accompanied by an increase in apoptosis;(f) is accompanied by a decrease in expression of Hoxa7, Hoxa9, andMeis-1; and/or (g) reduces leukemic engraftment of t(4;11)-positivecells.
 19. A double-stranded iRNA agent wherein the iRNA agent comprisesa sense strand and an antisense strand each 15-30 nucleotides in lengthand a double stranded region 15-30 basepairs in length and the antisensestrand comprises a region of complementarity to a mRNA encoding anMLL-AF4 fusion gene, wherein the region of complementarity is less than30 nucleotides in length, and the antisense strand comprises at least 15contiguous nucleotides from an antisense sequence of agents 1-12provided in Table 1 (SEQ ID NOs 6, 7, 11, 13, 15, 19, 21, 23, 25, 27,31, and 33).
 20. The iRNA agent of claim 19, selected from the groupconsisting of agents 1-12 provided in Table 1.